For the past two years now, teams have been able to participate in the InterLab Study to collect results of fluorescent measurements from all over the world. This year, the goal is to quantify the expression of five reporter constructs with GFP. All teams use three devices with different promoters and a GFP gene which was included in the distributed iGEM Kit. To conduct the experiments, we followed two protocols for the Plate Reader and the Flow Cytometer.
To start the study, the three given devices from iGEM, a negative and a positive control were transformed into E. coli TOP10 cells. Afterward, the transformed cells were incubated at 37 °C in LB medium.
Then, the colonies from each plate were picked and incubated overnight at 37 °C and 220 rpm in 10 ml medium with Chloramphenicol.
|Device 1||J23101 + I3504|
|Device 2||J23106 + I3504|
|Device 3||J23117 + I3504|
To be able to compare our results with other teams, LUDOX-S30 was used as a single point reference. 100 µl LUDOX and 100 µl H2O were pipetted into the wells following the scheme provided by iGEM HQ. The absorbance at 600 nm was measured of all samples in standard measurement modes.
In addition, a dilution series of FITC was measured in a 96-well plate in the plate reader. This generated a standard curve for FITC concentration, which can be converted into GFP concentration.
First, the FITC stock tube was spun down and a 2x FITC stock solution was produced by resuspending FITC in PBS. This solution was then incubated for 4 hours at 42 °C and afterward diluted with PBS to achieve a 250 µM concentration of the FITC stock solution. This solution was used to prepare the serial dilution of FITC. All samples were pipetted into the 96-well plate and lastly the fluorescence was measured.
The OD of the overnight culture was measured. When it reached an OD600 of 0.02, the cells were incubated for six hours in 10 ml 0,5x TB medium with Chloramphenicol at 37 °C and 220 rpm. A 100 µl sample was taken every hour. A measurement of the fluorescence (Abs600) was executed in 96-well plates following the protocol issued by the iGEM HQ.